Abstract Large-scale epidemiological and interventional studies have consistently demonstrated that diet and exercise have beneficial effects on mortality and lifespan, specifically they are associated with an increase in lifespan of diverse model organisms and decrease in human mortality from various diseases including heart disease, diabetes, stroke, cancer and neurodegenerative disorders. Many physiological changes in response to diet and exercise have been identified, however, it is still unknown which specific changes in metabolism drive the beneficial effects of diet and exercise on lifespan and age-associated diseases. At a cellular level, the key response to dietary manipulations and exercise involves changes in intracellular metabolic parameters such as ATP/ADP, NADH/NAD+, NADPH/NADP+, GSH/GSSG ratios and mitochondrial membrane potential. The causal relationship between changes in these crucial parameters and downstream effects of diet and exercise is currently unknown. A key bottleneck in understanding the role of intracellular metabolic parameters in regulation of metabolism has been the lack of methods for direct manipulation of these parameters in vivo. To fill this methodological gap, I have developed two genetically encoded tools for manipulation of NADH/NAD+ and NADPH/NADP+ ratios in living cells. Here, I propose to build on my expertise to develop a suite of genetically encoded tools for tissue- and compartment-specific manipulation of ATP/ADP, NADH/NAD+, NADPH/NADP+, GSH/GSSG ratios and mitochondrial membrane potential in living organisms. I will apply these tools to study the role of intracellular metabolic parameters in mediating the beneficial effects of calorie restriction, regulating energy metabolism and communicating intracellular energy status between cells. The availability of these tools will for the first time allow the research community to study the function of compartment- and tissue-specific changes in a particular metabolic parameter that is observed with diet, exercise or another physiological process. Understanding the role of intracellular metabolic parameters in mediating the beneficial effects of diet and exercise has the potential to transform our understanding of the effect of diet and exercise on aging and disease, which will lead to novel treatment and prevention approaches for common age-associated disorders.